2,3,5-trimethyl-4-hydroxyanilide derivatives, preparation thereof and therapeutical use thereof

ABSTRACT

Novel 2,3,5-trimethyl-4-hydroxyanilide derivatives of general formula I, wherein, e.g., R 1  is phenyl, R 2  is H, R 3  is C 12  H 25 , and A is a sulphur atom, are disclosed. A method for preparing said derivatives, pharmaceutical compositions containing at least one of said compounds as active principle, and the use of such derivatives for treating hypercholesterolemia or atherosclerosis. ##STR1##

The present application is a U.S. National Application filed under 35USC 371 of PCT/FR96/01877, filed Nov. 27, 1996 based upon Frenchapplication Ser. No. 95/14086 filed Nov. 28, 1995.

The subject-matter of the present invention is novel anilidederivatives, their preparation and their application in humantherapeutics.

It also relates to the use of these derivatives in the manufacture ofmedicaments intended for the treatment of hypercholesterolemia or ofatherosclerosis.

Dietary cholesterol is absorbed in the form of free cholesterol byintestinal cells and then esterified by the enzyme ACAT (acyl-CoA:cholesterol O-acyltransferase) in the serum. Inhibition of ACAT preventsthe intestinal absorption and the accumulation of cholesterol inarterial tissue. In addition, low density lipoproteins (LDL) are, afteroxidation, captured by scavenger receptors and result in the formationof the foam cell, the site of initiation of the atheromatous plaque (D.Steinberg et al., England. J. Med., 320, 915-924, 1989).

The object of the present invention is targeted at obtaining novelhypocholesterolemic and antioxidant derivatives which can act both onthe amount and the quality of the LDL, with the aim of reducing theiratherogenic potential and their long-term deleterious effects on thevascular wall.

The compounds of the present invention correspond to the general formulaI ##STR2## in which:

R₁ and R₂, which are identical or different, represent, independently ofone another:

hydrogen

a linear or branched C₁ -C₆ alkyl radical

an aromatic group, such as phenyl, naphthyl or pyridyl, optionallysubstituted by one or more C₁ -C₄ alkyl, C₁ -C₄ alkoxy, hydroxyl or halogroups

R3 represents a linear or branched C₆ -C₁₅ alkyl chain or a phenyl groupoptionally substituted by one or more C₁ -C₄ alkyl, C₁ -C₄ alkoxy,hydroxyl or halo groups

A represents an oxygen or sulfur atom or the sulfoxy group.

As it is possible for the compounds of general formula I to possessasymmetric centers, the present invention covers the variousstereoisomers or enantiomers and their mixtures.

The compounds of general formula I can be used in the preparation ofpharmaceutical compositions or of medicaments intended for the treatmentof diseases such as hypercholesterolemia or atherosclerosis.

Finally, the synthetic processes which make it possible to access thecompounds of general formula I also form part of the present invention.

The compounds of general formula I can be obtained according to one ofthe following methods (Scheme I):

Method A:

a) Treatment of 2,3,6-trimethyl-4-aminophenol hydrochloride with anα-haloacyl halide II, in which Hal and Hal' represent bromine orchlorine and R₁ and R₂ have the same meaning as above, in the presenceof a base, such as triethylamine, in order to access the compound III.

b) Treatment of the compound III with the derivative IV, in which R₃ andA have the same meaning as above, in a sodium/methanol or potassiumtert-butoxide/tert-butanol medium, in order to give the compound I.

Method B:

Treatment of 2,3,6-trimethyl-4-aminophenol hydrochloride with an α-haloacid V, in which Hal, R₁ and R₂ have the same meaning as above, in thepresence of an activator, such as dicyclohexylcarbodiimide or2-chloro-1-methylpyridinium iodide, and of a base, such astriethylamine, in order to access the compound III, subsequently treatedin a way identical to that described in Method A-b.

Method C:

Treatment of 2,3,6-trimethyl-4-aminophenol hydrochloride with thederivative IV, in which R₁, R₂, R₃ and A have the same meaning as above,in the presence of an activator, such as dicyclohexylcarbodiimide or2-chloro-1-methylpyridinium iodide, and of triethylamine, in order togive the compound I. ##STR3##

The invention can be better understood using the following non-limitingexamples which constitute advantageous embodiments according to theinvention.

EXAMPLE 1

(Method A)2',3',5'-Trimethyl-4'-hydroxy-α-(dodecylthio)propionanilide 1. ##STR4##a--2',3',5'-Trimethyl-4'-hydroxy-α-bromopropionanilide 1a.

Triethylamine (3.48 ml; 0.25 mol) is added to a solution of2,3,6-trimethyl-4-aminophenol hydrochloride (1.87 g; 0.01 mol) indimethylformamide placed under nitrogen. α-Bromopropionyl chloride (1.32ml; 0.0125 mol) is subsequently added dropwise and the reaction mixtureis stirred for one hour at room temperature.

After diluting with water, extraction is carried out with ethyl acetate.The organic phase is washed with N hydrochloric acid and with water andthen dried (MgSO₄) and concentrated to dryness under vacuum. The residueis taken up in hexane, filtered off and dried to give the compound 1a(2.10 g).

M.p.=186° C. TLC: silica gel 60F254 Merck Rf=0.61 (AcOEt).

b--2',3',5'-Trimethyl-4'-hydroxy-α-(dodecylthio)propionanilide 1.

n-Dodecanethiol (2.11 ml; 0.0088 mol) is dissolved in methanol (20 ml)and then sodium methoxide (0.47 g; 0.0088 mol) is added. After a contacttime of 15 minutes, the compound 1a (2.10 g; 0.0073 mol) is added andthe reaction mass is brought to 60° C. for 2 hours. The methanol issubsequently evaporated off and then the residue is extracted with ethylacetate.

The organic phase, washed with water and then dried (MgSO₄), isconcentrated to dryness under vacuum. The residue obtained is purifiedby flash chromatography (elution: 30/70 ethyl acetate/hexane) to give1.24 g of white crystals (1).

M.p.=123° C. TLC: silica gel 60F254 Merck Rf=0.59 (50/50 AcOEt/hexane).

EXAMPLE 2

2',3',5'-Trimethyl-4'-hydroxy-α-(dodecylthio)acetanilide 2. ##STR5##

This compound is prepared according to the process described in Example1, by using bromoacetyl bromide.

M.p.=99° C. TLC: silica gel 60F254 Merck Rf=0.51 (50/50 AcOEt/hexane).

EXAMPLE 3

2',3',5'-Trimethyl-4'-hydroxy-α-(dodecylthio)butyranilide 3. ##STR6##

This compound is prepared according to the process described in Example1, by using 2-bromobutyryl bromide.

M.p.=127° C. TLC: silica gel 60F254 Merck Rf=0.61 (50/50 AcOEt/hexane).

EXAMPLE 4

2',3',5'-Trimethyl-4'-hydroxy-α-(dodecylthio)hexananilide 4. ##STR7##

This compound is prepared according to the process described in Example1, by using 2-bromohexanoyl bromide.

M.p.=80° C. TLC: silica gel 60F254 Merck Rf 0.36 (30/70 AcOEt/hexane).

EXAMPLE 5

2',3',5'-Trimethyl-4'-hydroxy-α-(dodecylthio)isovaleranilide 5. ##STR8##

This compound is prepared according to the process described in Example1, by using 2-bromoisovaleryl chloride.

M.p.=123° C. TLC: silica gel 60F254 Merck Rf=0.30 (30/70 AcOEt/hexane).

EXAMPLE 6

2',3',5'-Trimethyl-4'-hydroxy-α-(dodecylthio)valeranilide 6. ##STR9##

This compound is prepared according to the process described in Example1, by using 2-bromovaleryl bromide. M.p.=116° C. TLC: silica gel 60F254Merck Rf=0.39 (30/70 AcOEt/hexane).

EXAMPLE 7

(Method B)2',3',5'-Trimethyl-4'-hydroxy-α-dodecylthio-α-phenylacetanilide 7.##STR10## a)--2',3',5'-Trimethyl-4'-hydroxy-α-chloro-α-phenylacetanilide7a.

Triethylamine (0.94 ml; 0.0067 mol) is added to a suspension of2,3,6-trimethyl-4-aminophenol hydrochloride (1.27 g; 0.0067 mol) inmethylene chloride (35 ml) placed under nitrogen.

α-Chlorophenylacetic acid (1.27 g; 0.0074 mol) anddicyclohexylcarbodiimide (1.54 g; 0.0074 mol) are subsequently added andthe reaction mixture is vigorously stirred for 2 hours at roomtemperature.

After filtering off the dicyclohexylurea formed, the organic phase iswashed with N/10 hydrochloric acid, with water and then with aqueoussaline solution. After drying (MgSO₄) and evaporating to dryness undervacuum, the residue is taken up in ethyl ether. The crystals formed arefiltered off and dried to give the compound 7a (1.22 g).

M.p.=199° C. TLC: silica gel 60F254 Merck Rf=0.68 (50/50 AcOEt/hexane).

b)--2',3',5'-Trimethyl-4'-hydroxy-α-dodecylthio-α-phenylacetanilide 7.

The compound is prepared according to the technique described in Example1b, starting from the compound 7a.

M.p.=129° C. TLC: silica gel 60F254 Merck Rf=0.64 (50/50 AcOEt/hexane).

EXAMPLE 8

(Method C) 2',3',5'-Trimethyl-4'-hydroxy-α-(dodecylthio)isobutyranilide8. ##STR11##

2,3,6-Trimethyl-4-aminophenol hydrochloride (3.52 g; 0.018 mol),α-(dodecylthio)isobutyric acid (5.41 g; 0.018 mol) and triethylamine(9.4 ml; 0.067 mol) are successively added to a suspension of2-chloro-1-methylpyridinium iodide (5.75 g; 0.022 mol) in chloroform(225 ml) and then the reaction mixture is heated at reflux for 2 hours.The reaction mass is cooled, diluted with ethyl ether (350 ml) and thenfiltered. This organic phase is subsequently washed with N hydrochloricacid, with water and then with aqueous saline solution. After drying(MgSO₄) and concentrating to dryness under vacuum, the residue is takenup in isopropyl ether and filtered off to give 7.32 g of white crystalsof the compound 8.

M.p.=71° C. TLC: silica gel 60F254 Merck Rf=0.65 (50/50 AcOEt/hexane).

EXAMPLE 9

2',3',5'-Trimethyl-4'-hydroxy-α-(p-chlorophenylthio)isobutyranilide 9.##STR12##

This compound is prepared according to the process described in Example8, by using α-(p-chlorophenylthio)isobutiric acid.

M.p.=134° C. TLC: silica gel 60F254 Merck Rf=0.54 (50/50 AcOEt/hexane).

EXAMPLE 10

2',3',5'-Trimethyl-4'-hydroxy-α-(p-chlorophenylsulfinyl)isobutyranilide10. ##STR13##

This compound is prepared according to the process described in Example8, by using α-(p-chlorophenylsulfinyl)isobutyric acid.

M.p.=157-158° C. TLC: silica gel 60F254 Merck Rf=0.33 (50/50AcOEt/hexane).

EXAMPLE 11

2',3',5'-Trimethyl-4'-hydroxy-α-(p-chlorophenoxy)isobutyranilide.##STR14##

Ethyl chloroformate (0.96 ml; 0.01 mol) is added dropwise to a solution,cooled to 0° C., of clofibric acid (2.14 g; 0.01 mol) and oftriethylamine (1.48 ml; 0.0105 mol) in tetrahydrofuran (25 ml). Afterstirring for 20 minutes, the mixed anhydride obtained is added slowly toa suspension of 2,3,6-trimethyl-4-aminophenol hydrochloride (1.87 g;0.01 mol) in dimethylformamide (10 ml) and triethylamine (1.48 ml;0.0105 mol).

The reaction mixture, kept under a nitrogen stream, is stirred for 1hour at 5° C. and then for 12 hours at room temperature, then pouredinto water and extracted with ethyl acetate. The organic phase is washedwith water and with aqueous saline solution, dried over MgSO₄ and thenevaporated to dryness under vacuum. The residue is crystallized fromethyl ether and then recrystallized from ethyl acetate to give thecompound 11.

M.p.=175° C. TLC: silica gel 60F254 Merck Rf=0.30 (30/70 AcOEt/hexane).

EXAMPLE 12

2',3',5'-Trimethyl-4'-hydroxy-α-(dodecylsulfinyl)bobutyranilide 12.##STR15##

This compound is prepared according to the process described in Example8, by using α-(dodecylsulfinyl)isobutyric acid.

M.p.=73° C. TLC: silica gel 60F254 Merck Rf=0.43 (AcOEt/hexane).

EXAMPLE 13

2',3',5'-Trimethyl-4'-hydroxy-α-dodecylthio-α-3,5-di-tert-butyl-4-hydroxyphenylacetanilide13. ##STR16##

This compound is prepared according to the method described in Example11, by using α-dodecylthio-3,5-di-tert-butyl-4-hydroxyphenylacetic acid.

M.p.=150° C. TLC: silica gel 60F254 Merck Rf=0.31 (30/70 AcOEt/hexane).

EXAMPLE 14

2',3',5'-Trimethyl-4-hydroxy-α-dodecylthio-α-(p-methoxyphenyl)acetanilide14. ##STR17##

This compound is prepared according to the method described in Example7a, by using α-dodecylthio-α-(p-methoxyphenyl)acetic acid.

M.P.=122° C. TLC: silica gel 60F254 Merck Rf=0.74 (30/70 AcOEt/hexane).

EXAMPLE 15

2',3',5'-Trimethyl-4'-hydroxy-α-dodecylthio-α-naphthylacetanilide 15.##STR18##

This compound is prepared according to the method described in Example7a, by using α-dodecylthio-α-naphthylacetic acid.

M.p.=134° C. TLC: silica gel 60F254 Merck Rf: 0.60 (95/5 CH₂ Cl₂/AcOEt).

EXAMPLE 16

(+)-2',3',5'-Trimethyl-4'-hydroxy-α-dodecylthio-α-phenylacetanilide 16.##STR19##

This compound is prepared according to the method described in Example7a, by using (+)-α-dodecylthio-α-phenylacetic acid.

M.p.=128° C. TLC: silica gel 60F254 Merck Rf=0.64 (50/50 AcOEt/hexane).α_(D) ²⁵ =+34.7° (C=0.5; ethanol).

The compounds of the invention were subjected to pharmacological testswhich showed their potential value in the treatment ofhypercholesterolemia and in the treatment of atheromatous disease.

The compounds were studied for their inhibitory effect on ACAT andhypocholesterolemic effect in rats, on the one hand, and for theirantioxidant effect, on the other hand.

1) Inhibition of ACAT

The inhibitory activity of the compounds with respect to ACAT(acyl-CoA:cholesterol O-acyl-transferase enzyme) was evaluated in vitrousing the technique of H. Chautan et al. (Analytical Biochemistry, 173,436-439, 1988).

The activities, expressed as 50% inhibitory concentrations (IC₅₀),obtained with some products of the invention are recorded, by way ofexample, in Table 1 below:

                  TABLE 1                                                         ______________________________________                                        Compounds No.   IC.sub.50 (μM)                                             ______________________________________                                        2               0.30                                                          3               0.31                                                          4               0.16                                                          5               0.63                                                          6               0.11                                                          7               0.18                                                          8               0.19                                                          9               1.12                                                          11              1.10                                                          16              0.16                                                          CI 976          1.04                                                          DUP 128         0.1                                                           ______________________________________                                    

2) Hypocholesterolemic activity

Male rates (160-180 g) are subjected for 4 days to an Altromin C 1061hypercholesterolemic diet, and concomitantly treated orally withcompounds suspended in a 2% solution of Tween 80 in distilled water.

On day 5, the animals, which have not fasted, are anesthethized withethyl ether and exsanguinated by drawing blood at the abdominal aortaonto EDTA. The blood is immediately centrifuged and the plasma stored at4° C.

Plasma cholesterol is then assayed by the CHOD-PAP method(Boehringer-Mannheim Ref. 237574). The median effective dose (ED₅₀)corresponds to the dose which reduces the plasma cholesterolconcentration by half relative to control animals.

    ______________________________________                                        Compounds No.  ED.sub.50 (mg/kg)                                              ______________________________________                                        2              >10                                                            3              4                                                              4              0.5                                                            5              1                                                              6              1                                                              7              0.2                                                            8              10                                                             14             1                                                              16             0.15                                                           CI 976         8.3                                                            DUP 128        1.1                                                            ______________________________________                                    

3) Antioxidant activity

a) Chemical peroxidation.

In the presence of Fe³⁺ and ADP, dihydroxyfumaric acid undergoes anautoxidation which generates oxygen free radicals. The latter bringabout the peroxidation of hepatic microsomal lipids.

This peroxidation, performed on rat liver microsomes, is measuredaccording to the thiobarbituric acid technique (formation of TBARS), asdescribed by S. Y. H. Tse et al. (Biochemical Pharmacology, Vol. 42, No.3, 459-464, 1991).

    ______________________________________                                        Compounds No.   IC.sub.50 (μM)                                             ______________________________________                                        2               5                                                             3               >10                                                           4               0.5                                                           5               5                                                             6               0.3                                                           7               0.6                                                           8               3                                                             CI 976          >10                                                           DUP 128         >10                                                           Vitamin E       2.3                                                           ______________________________________                                    

b) Oxidation of LDL.

Human LDL (Sigma L 2139) are oxidized with 10 μM CuSO₄. After anincubation period of 6 hours, the peroxidation is evaluated by measuringthe TBARS by spectrophotometry at 532 nanometers.

    ______________________________________                                        Compounds No.   IC.sub.50 (μM)                                             ______________________________________                                         4              10                                                             7              13                                                            12               3                                                            16               4                                                            CI 976          100                                                           DUP 128         30                                                            Vitamin E       10                                                            ______________________________________                                    

The compounds of the invention are hypocholesterolemic agents thatinhibit ACAT and antioxidants, which can be used for the treatment ofdiseases such as hypercholesterolemia and atherosclerosis.

The pharmaceutical compositions can be presented in the form appropriatefor oral, parenteral or local administration, for example in the form ofcapsules, including hard gelatin capsules, tablets, granules, liquidsolutions, syrups or suspensions to be swallowed, and can contain theappropriate excipients.

The daily dosage can range from 10 to 3000 mg.

We claim:
 1. An anilide derivative, selected from those of generalformula I ##STR20## in which: R₁ and R₂, which are identical ordifferent, represent, independently of one another:hydrogen linear orbranched C₁ -C₆ alkyl an aromatic group, selected from the groupconsisting of phenyl, naphthyl, and pyridyl, optionally substituted byone or more C₁ -C₄ alkyl, C₁ -C₄ alkoxy, hydroxyl, or halo; R3represents linear or branched C₆ -C₁₅ alkyl or phenyl optionallysubstituted by one or more C₁ -C₄ alkyl, C₁ -C₄ alkoxy, hydroxyl orhalo; A represents oxygen or sulfur or sulfoxy;and its variousstereoisomers or enantiomers, and their mixtures, for the a compoundexhibiting one or more asymmetric centers.
 2. A compound according toclaim 1, selected from thefollowing:2',3',5'-trimethyl-4'-hydroxy-α-(dodecylthio)propionanilide,2',3',5'-trimethyl-4'-hydroxy-α-(dodecylthio)acetanilide,2',3',5'-trimethyl-4'-hydroxy-α-(dodecylthio)butyranilide, ' .3',5'-trimethyl-4'-hydroxy-α-(dodecylthio)hexananilide,2',3',5'-trimethyl-4'-hydroxy-α-(dodecylthio)isovaleranilide,2',3',5'-trimethyl-4'-hydroxy-α-(dodecylthio)valeranilide,2',3',5'-trimethyl-4'-hydroxy-α-dodecylthio-α-phenylacetanilide2',3',5'-trimethyl-4'-hydroxy-α-(dodecylthio)isobutyranilide,2',3',5'-trimethyl-4'-hydroxy-α-(p-chlorophenylthio)isobutyranilide,2',3',5'-trimethyl-4'-hydroxy-α-(p-chlorophenylsulfinyl)isobutyranilide,2',3',5'-trimethyl-4'-hydroxy-α-(p-chlorophenoxy)isobutyranilide,2',3',5'-trimethyl-4'-hydroxy-α-(dodecylsulfinyl)bobutyranilide,2',3',5'-trimethyl-4'-hydroxy-α-dodecylthio-α-(3,5-di-tert-butyl-4-hydroxy)phenylacetanilide,2',3',5'-trimethyl-4'-hydroxy-α-dodecylthio-α-(p-methoxyphenyl)acetanilide,2',3',5'-trimethyl-4'-hydroxy-α-dodecylthio-α-naphthylacetanilide, and(+)-2',3',5'-trimethyl-4'-hydroxy-α-dodecylthio-α-phenylacetanilide. 3.Process for the preparation of a compound according to claim 1,wherein:a) in a first stage, 2,3,6-trimethyl-4-aminophenol hydrochlorideis treated with an α-haloacyl halide in the presence of a base, in orderto provide the intermediate III ##STR21## in which R₁ and R₂ are asdefined in claim 1 and Hal represents a chlorine or bromine atom; b) ina second stage, the intermediate III is treated with a derivative R₃(A)H, in which R₃ and A are as defined in claim 1, in a sodium/methanolor potassium tert-butoxide/tert-butanol medium.
 4. Process for thepreparation of a compound according to claim 3, wherein the intermediateIII is alternatively obtained by reaction of an α-halo acid with2,3,6-trimethyl-4-aminophenol hydrochloride in the presence of anactivator, and of a base.
 5. Process for the preparation of a compoundaccording to claim 1, characterized in that2,3,6-trimethyl-4-aminophenol hydrochloride is reached with a derivativeVI ##STR22## in which R₁, R₂, R₃ and A are as defined in claim 1,activation being carried out with ethyl chloroformate or withdicyclohexylcarbodiimide or 2-chloro-1-methylpyridinium iodide in thepresence of a base.
 6. A pharmaceutical composition comprising inaddition to a pharmaceutically-acceptable vehicle, at least one compoundof claim
 1. 7. A pharmaceutical composition comprising, in addition to apharmaceutically-acceptable vehicle, at least one compound of claim 2.8. A method for treatment of hypercholesterolemia or atherosclerosiscomprising the step of administering, to a living body suffering fromthe same, an effective amount of a compound of claim
 1. 9. A method fortreatment of hypercholesterolemia or atherosclerosis comprising the stepof administering, to a living body suffering from the same, an effectiveamount of a compound of claim
 2. 10. A process of claim 3 wherein thebase is triethylamine.
 11. A process of claim 4 wherein the activator isdicyclohexylcarbodiimide or 2-chloro-1-methylpyridinium iodide and thebase is triethylamine.
 12. A process of claim 5 wherein the base istriethylamine.